Impact of Formulation Conditions on Lipid Nanoparticle Characteristics and Functional Delivery of CRISPR RNP for Gene Knock-Out and Correction

The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse genetic disorders. However, for gene therapy applications, an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. In this study, we optimized the formulation conditions of lipid nanoparticles (LNP) for delivery of ready-made CRISPR-Cas9 ribonucleic protein (RNP). The buffer composition during complexation and relative DOTAP concentrations were varied for LNP encapsulating in-house produced Cas9 RNP alone or Cas9 RNP with additional template DNA for gene correction. The LNP were characterized for size, surface charge, and plasma interaction through asymmetric flow field flow fractionation (AF4). Particles were functionally screened on fluorescent reporter cell lines for gene knock-out and gene correction. This revealed incompatibility of RNP with citrate buffer and PBS. We demonstrated that LNP for gene knock-out did not necessarily require DOTAP, while LNP for gene correction were only active with a low concentration of DOTAP. The AF4 studies additionally revealed that LNP interact with plasma, however, remain stable, whereby HDR template seems to favor stability of LNP. Under optimal formulation conditions, we achieved gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively, at nanomolar concentrations of the CRISPR-Cas9 RNP

Weekly high-dose cisplatin is a feasible treatment option: Analysis on prognostic factors for toxicity in 400 patients

In the present study we describe the toxicity of weekly high-dose (70-85 mg m-2) cisplatin in 400 patients (203 men, 197 women; median age 54 years) with advanced solid tumours treated in the period 1990-2001 who took part in phase I/II trials, investigating the feasibility and efficacy of weekly cisplatin alone, or in combination with paclitaxel or etoposide. Cisplatin was administered in 250 ml NaCl 3% over 3 h, for six intended administrations. The mean number of administrations was 53 (range, 1-6 administrations). Reasons not to complete six cycles were disease progression (7.5%), haematological toxicity (9%), nephrotoxicity (7%), ototoxicity (2.5%), neurotoxicity (1%), gastrointestinal toxicity (1%), cardiovascular complications (0.5%) or a combination of reasons including noncompliance and patient's request (5.5%). Logistic regression analysis was used to evaluate baseline parameters for prognostic value regarding toxicity. Leukopenia correlated with etoposide cotreatment, and thrombocytopenia with cisplatin dose and prior (platinum-based) chemotherapy. Risk factors for nephrotoxicity were older age, female gender, smoking, hypoalbuminaemia and paclitaxel coadministration. Neurotoxicity >grade I (11% of patients) was associated with prior chemotherapy and paclitaxel coadministration. Symptomatic hearing loss occurred in 15% with anaemia as the predisposing factor. We conclude that weekly high-dose cisplatin administered in hypertonic saline is a feasible treatment regimen

Using a satellite swarm for building a space-based radio telescope for low frequencies

In radio astronomy, as in astronomy in general, a wide range of frequencies is observed as each spectral band o_ers a unique window to study astrophysical phenomena. In the recent years, new observatories have been designed and built at the extreme limits of the radio spectrum. For the low frequencies several Earth-based radio telescopes are constructed at this moment. In the Netherlands, the Low Frequency Array (LOFAR) is being constructed at this moment and will be operational later this year. LOFAR observes the sky between 30 and 240 MHz. Observing at even lower frequencies is very interesting, but, due to the inuence of the Earth's ionosphere this is not possible from Earth. Thus, the only option to observe low frequencies is a telescope in spac

OLFAR - orbiting low frequency array; using a satellite swarm for building a space-based radio telescope for low frequencies

In radio astronomy, as in astronomy in general, a wide range of frequencies is observed as each spectral band offers a unique window to study astrophysical phenomena. In the recent years, new observatories have been designed and built at the extreme limits of the radio spectrum. For the low frequencies several Earth-based radio telescopes are constructed at this moment. In the Netherlands, the Low Frequency Array (LOFAR) is being constructed at this moment and will be operational later this year. LOFAR observes the sky between 30 and 240 MHz. Observing at even lower frequencies is very interesting, but, due to the influence of the Earth’s ionosphere this is not possible from Earth. Thus, the only option to observe low frequencies is a telescope in space

Model-based analyses: Promises, pitfalls, and example applications to the study of cognitive control

We discuss a recent approach to investigating cognitive control, which has the potential to deal with some of the challenges inherent in this endeavour. In a model-based approach, the researcher defines a formal, computational model that performs the task at hand and whose performance matches that of a research participant. The internal variables in such a model might then be taken as proxies for latent variables computed in the brain. We discuss the potential advantages of such an approach for the study of the neural underpinnings of cognitive control and its pitfalls, and we make explicit the assumptions underlying the interpretation of data obtained using this approach